The long-term goal of this program is understanding the basis for regulation of ATP-sensitive potassium KATP channels by nucleotides and pharmacologic agents. KATP channels couple membrane electrical activity to cellular energy metabolism and are key regulators in physiologic processes ranging from control of glucose stimulated insulin release to maintenance of vascular smooth muscle tone. Loss of beta-cell KATP channels is a cause of congenital hyperinsulinism, while sulfonylureas like tolbutamide and glibenclamide, are a mainstay in the treatment of NIDDM. Sulfonylurea receptors, SURs, associate with and regulate the pore forming KIR subunits of KATP channels. Past work has established the stoichiometry of KATP channels, (KIR6.x/SUR)4, and identified SUR domains which specify channel isoform differences including those critical for the action of channel blockers and openers. Without SURs, homomeric KIR6.2 pores have a low open channel probability, POmax, are insensitive to sulfonylureas, display altered bursting, are poorly inhibited by ATP, and not stimulated by MgADP, all properties reversed by co-assembly with an SUR. Recent work demonstrates that the first set of transmembrane helices, TMD0, and a connecting segment of SUR, L0, and the amino terminus of KIR6.x, are critical for the control of slow gating. These segments affinity-label with analogues of glibenclamide arguing for their close proximity. The SUR domain critical for SUR-KIR assembly has been identified. TMD0 interacts with KIRS increasing their Pomax while L0 modulates gating in a bi-directional manner. The goal of the proposed research is to define the inter- and intramolecular interactions involved in the assembly and regulation of KATP channels. We have three specific aims: (1) To define the minimal segment of TMD0 needed to interact with KIR6.2 and determine if this is sufficient to activate (KIR6.2deltaC)4 pores. (2) To test the hypothesis that the amino terminus of KIR6.x interacts with the L0 linker of SURs to control bursting. (3) To define further the sulfonylurea/glibenclamide binding pocket of SUR1, specifically to identify amino acids in proximity to the meglitinide head group.